Mutans streptococci have been convincingly implicated in the initiation of dental caries in humans. The ability of these organisms to accumulate and colonize on the tooth surface has been associated with the synthesis of glucans from sucrose. Glucans are synthesized by constitutively secreted glucosyltransferase (GTF) enzymes. These enzymes have been considered as potential components of a dental caries vaccine because of their role in the pathogenicity of Mutans streptococci. However, vaccines based on intact GTF have a variety of disadvantages, such as the presence of inappropriate epitopes and excess molecular material that does not possess appropriate immunogenic features.
It is quite likely that protection against dental caries will involve functional inhibition of the catalytic and/or the glucan-binding activities of GTF. Epitopes associated with these functions would theoretically be primary targets for immunological attack, provided that the relevant sequences are located in molecular areas that are accessible to antibody. Subunit vaccines provide a method to block functional domains without inducing immunity to irrelevant or unwanted epitopes. It has been reported that synthetic peptide vaccines associated with catalytic or glucan-binding domains of GTF can protect rats from experimental dental caries (Taubman et al., Infect. Immun. 63:3088-3093 (1995)). One of the peptides that was successfully used as a vaccine (Smith et al., Infect. Immun. 62:5470-5476 (1994)) demonstrated a sequence containing an aspartic acid (aspartate 451 in S. mutans GTF-B) to which the glucosyl moiety of sucrose was covalently bound (Mooser et al., J. Biol. Chem. 266:8916-8922 (1991)).
Mutans streptococcal GTFs may contain several sequentially separated residues that, partly because of secondary structure constraints (e.g., (xcex2,xcex1)8 barrel domain), have important functions in GTF catalytic mechanisms. Peptides containing catalytically implicated aspartates (Asp 413/415 or Asp 451, based on the sequence numbering of S. mutans GTF B) each induced antibody that inhibited GTF activity and protected rats from experimental dental caries. Recent site-directed mutagenesis and comparative sequence studies have implicated these sequentially separated residues in the catalytic activity of mutans streptococcal glucosyltransferases (GTF). The immunogenicity and induction of GTF-inhibitory activity of synthetic peptides which contained putative catalytic regions that were associated with the xcex25 (EAW peptide) and xcex27 (HDS peptide) strand elements of the suggested (xcex2,xcex1)8 catalytic barrel domain of GTF were examined. Both peptides induced serum IgG and salivary IgA anti-peptide activity twenty one days after the second injection. Serum IgG antibody induced by HDS and EAW peptide constructs also showed significant reaction with S. mutans GTF. Antisera to each peptide construct also inhibited the ability of S. mutans GTF to synthesize glucan. These observations support the existence of catalytic subdomains containing glutamate and tryptophan (EAW) or aspartate and histidine (HDS) residues which have been suggested to be involved with the catalytic activity of GTF. Furthermore, the epitope(s) defined in these sequences have significant immunogenicity and can induce immune responses which interfere with GTF-mediated glucan synthesis.
This invention pertains to subunit vaccine compositions which elicit immune system responses in mammals to glucosyltransferase (GTF), an enzyme that is implicated in the formation of dental caries, or to subunits thereof. Rather than using intact GTF as an immunizing agent, the vaccine composition or immunogenic composition is prepared from particular immunogenic portions (subunits) of GTF.
The invention relates to vaccine compositions and immunogenic compositions comprising at least one peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered. In particular embodiments, the amino acid sequence is selected from the group consisting of 481-ANDHLSILEAWSDNDTPYLHD (EAW peptide; SEQ ID NO: 1); and 552-VPSYSFIRAHDSEVQDLIA (HDS peptide; SEQ ID NO: 2). These peptides are believed to be from the catalytic domain of GTF and have been shown to induce high levels of antibody that crossreact with intact GTF.
In another embodiment, the invention relates to a peptide having the amino acid sequence 1300-TGARTINGQLLYFRANGVQVKG (GLB peptide; SEQ ID NO: 3); this sequence is believed to be from the glucan binding region of GTF.
In a particularly preferred embodiment, 2 or more of the peptides are present and arranged on a core matrix of 3 or more lysines.
The invention also relates to vaccine compositions and immunogenic compositions comprising at least two peptides covalently attached to a peptidyl core matrix, wherein each peptide consists essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered. In a particular embodiment, the amino acid sequence is selected from the group consisting of the EAW peptide (ANDHLSILEAWSDNDTPYLHD; (SEQ ID NO: 1)) and the HDS peptide (VPSYSFIRADSEVQDLIA; (SEQ ID NO: 2)). In another embodiment, the peptide is the GLB peptide (TGARTINGQLLYFRANGVQVKG; (SEQ ID NO: 3)). In additional embodiments, the composition further comprises at least one additional immunologic component covalently attached to said peptidyl core matrix. For example, the additional immunologic component can be an immunogenic portion of a pathogen selected from the group consisting of diphtheria, pertussis, tetanus, measles and polio vaccines.
The invention also pertains to a method of provoking an immune response to glucosyltransferase in mammals comprising administering to a mammal at least one peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, which is of sufficient length to raise an immune response in the mammal, thereby provoking said immune response. In a preferred embodiment, the immune response results in reduction of the colonization or accumulation of mutans streptococcal strains in the mammal to whom the peptide is administered.
The invention further pertains to a method of immunizing a mammal against dental caries comprising administering to the mammal at least one peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in the mammal.